The slime mold Dictyostelium discoideum provides an exceptional experimental system for investigating thr regulation of developmental timing due to several unique timing features including: 1) the capacity to identify and characterize the individual rate-limiting components f developmental timers which cue the consecutive stages of morphogenesis; 2) the capacity to isolate a unique class of timer mutants in which individual rate-limiting components are either contracted, expanded, or in one case eliminated, without affecting the sequence of morphogenetic stages; 3) the capacity to stimulate by disaggregation the rapid recapitulation of morphogenesis, which then reoccurs in one-tenth the original time; 4) the capacity to stimulate a reverse program of "erasure" and dedifferentiation by simply disaggregating developing cells and resuspending them in nutrient medium, and 5) the capacity to stimulate parallel programs of dedifferentiation and redifferentiation in the same cell. We will continue to exploit these unique timing features in order to investigate the regulation of developmental timing and gene expression during the forward program of morphogenesis and the reverse program of erasure and didifferentiation. We propose to 1) investigate the relationship between the rate-limiting components of the preaggregative period and the regulation of associated gene expression, 2) isolate and characterize new timer mutants, 3) examine the newly discovered phenomenon of high frequency "switching" in timer mutants, 4) test whether transposable genetic elements are involved in "switching", 5) investigate the mechanisms involved in turning vegetative genes on and developmental genes off during the erasure process, and 6) continue to isolate and characterize new erasure-defective, or dedifferentiation-defective mutants. In the studies involving gene regulation, the programs of protein synthesis will be assessed by high resolution 2D-PAGE, and charges in the levels of specific mRNA's will be assessed by northern blots employing cloned DNA's containing developmentally regulated genes. Our ultimate goals are 1) to understand why morphogenetic events happen when they do andy why genes are turned on and off at exact times in a developmental program, 2) to test whether mobile genetic elements are involved in the regulation of developmental timing, and 3) to elucidate the mechanism of the "erasure event" which initiates the program of dedifferentiation.